university of copenhagen

University of Copenhagen

An efficient, robust, and inexpensive grinding device for herbal samples like Cinchona
bark
Hansen, Steen Honor; Holmfred, Else Skovgaard; Cornett, Claus; Maldonado Goyzueta,
Carla Brenda; Rnsted, Nina
Published in:
Scientia Pharmaceutica
DOI:
10.3797/scipharm.1410-14
Publication date:
2015
Document Version
Publisher's PDF, also known as Version of record
Citation for published version (APA):
Hansen, S. H., Holmfred, E. S., Cornett, C., Maldonado Goyzueta, C. B., & Rnsted, N. (2015). An efficient,
robust, and inexpensive grinding device for herbal samples like Cinchona bark. Scientia Pharmaceutica, 83(2),
369-376. DOI: 10.3797/scipharm.1410-14

Download date: 13. Sep. 2016

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Open Access

Research article

An Efficient, Robust, and Inexpensive Grinding

Device for Herbal Samples like Cinchona Bark
Steen Honor HANSEN * 1, Else HOLMFRED 1,
Claus CORNETT 1, Carla MALDONADO 2, Nina RNSTED 2
1
2

Analytical Biosciences, Dept. of Pharmacy, Fac. of Health and Med. Sci., Univ. of Copenhagen, Denmark.
Natural History Museum of Denmark, University of Copenhagen, Denmark.

* Corresponding author. E-mail: steen.honorehansen@sund.ku.dk (S. H. Hansen)

Sci Pharm. 2015; 83: 369376
Published:
Accepted:

th

February 19 2015
February 19th 2015

doi:10.3797/scipharm.1410-14
Received:

October 20th 2014

This article is available from: http://dx.doi.org/10.3797/scipharm.1410-14

Hansen et al.; licensee sterreichische Apotheker-Verlagsgesellschaft m. b. H., Vienna, Austria.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited.

Abstract
An effective, robust, and inexpensive grinding device for the grinding of herb
samples like bark and roots was developed by rebuilding a commercially
available coffee grinder. The grinder was constructed to be able to provide
various particle sizes, to be easy to clean, and to have a minimum of dead
volume. The recovery of the sample when grinding as little as 50 mg of crude
Cinchona bark was about 60%.
Grinding is performed in seconds with no rise in temperature, and the grinder is
easily disassembled to be cleaned. The influence of the particle size of the
obtained powders on the recovery of analytes in extracts of Cinchona bark was
investigated using HPLC.

Keywords
Grinding Bark and root Cinchona bark HPLC

Introduction
Sample preparation is an important first step in the chemical analysis of constituents in
herbs. Typically, sample preparation involves pulverization and liquid extraction processes.
The grinding of herbal samples like bark and roots into a fine powder is necessary in order
to obtain more homogeneous samples, which again will result in more reliable analytical
data. A more homogeneous sample obtained by pulverization also allows for the use of
less sample material for analysis.

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S. H. Hansen et al.:

Furthermore, larger particles or pieces of plant material may be difficult to moisten and
therefore, extract quantitatively. A finely ground sample is thus preferred for analytical
chemical investigations.
The grinding of herbal samples like bark and roots can be performed in several ways by
using a mortar and pestle, crushing after freezing in liquid nitrogen (~cryogenic grinding)
[1, 2], in a ball mill, or other kinds of milling machines [3]. When using the first method, it is
difficult to obtain a very fine and homogeneous powder and it is not possible to control the
particle size of the final powder. The two latter techniques require relatively expensive
equipment.
In the present paper, it is described how to modify a commercial coffee grinder into an
effective and robust device that is able to handle and grind bark or root samples as small
as 50 mg with a good recovery. The influence of the particle size on the extraction
efficiency is also investigated. Although larger amounts of a sample can be ground in this
device, the focus has been on the development of a device which could grind small
samples with a reasonable recovery to be used also with herbarium specimens where
possible sample sizes are limited. Additionally, the grinder is designed to be used for
analytical problems including the need for grinding hundreds of bark or root samples
requiring efficiency and speed in the grinding process.
The device is able to grind small bark samples of 0.05 g to 2.0 g within less than 10 sec
and with a recovery > 60% at the 0.05 g level.

Results and Discussion

The Grinder
The Rancilio coffee grinder was modified in order to minimize the dead volume as much as
possible. This was of utmost importance in order to be able to have a reasonably high
recovery when grinding samples as small as 50 mg. The cleaning of the grinder between
samples to avoid carryover is another important item. This is solved by adding a pin to the
upper cutter, which can then be disassembled within 10 sec.
The recovery of the ground sample was investigated by grinding different amounts of the
same Cinchona bark and by weighing the collected powder (Table 1).
It is seen that the recovery decreases with decreasing sample size. The main reason for
the loss is that some of the sample is retained in the gratings of the two grinding wheels,
which therefore have to be cleaned between samples in order to avoid carryover between
samples. Even at the level of 50 mg of crude bark, about 30 mg of milled bark is recovered
as a suitable powder for further analysis. For HPLC analysis, 100 mg of powder extracted
to a volume of 10 mL is used, but this sample preparation may easily be scaled down by a
factor of ten.

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A Robust and Inexpensive Grinding Device for Herbal Samples

Tab. 1.

371

Recovery of Cinchona bark after grinding

Sample
500 1
500 2
500 3
250 1
250 2
250 3
100 1
100 2
100 3
50 1
50 2
50 3

mg
before grinding
502.6
515.5
520.1
262.0
264.5
251.7
101.0
98.3
101.4
52.2
50.2
50.6

mg
after grinding
349.5
385.8
361.6
195.5
199.0
190.3
73.2
63.8
65.3
30.4
29.2
25.7

Recovery %

Recovery
Mean %

70
75
70
75
75
76
72
65
64
58
58
51

71

75

67

56

The important task is to obtain a fine powder without any larger particles. This will support
an increase in the repeatability of the measurements, whereas powder with small and
large particles together may result in less repeatable results when using small sampling
sizes. A number of powders with various particle size distributions obtained by grinding the
same Cinchona bark were analysed for the content of quinine by HPLC. For the extraction,
a standard extraction solvent of 70% methanol containing 0.1% formic acid was used. No
recovery studies were performed because only the effect of the particle size was to be
evaluated.
Tab. 2.

HPLC determination of quinine in Cinchona bark ground to four various particle

sizes. Three separate determinations are performed on each sample
Sample
Cinchona bark
1
2
3
4

Particle size
Range (m) Mean (m)
254000
680
152000
590
101000
400
101000
430

Peak area
(n=3)
2402; 2428; 2630
2845; 3063; 2892
3432; 3698; 3552
3700; 3615; 3478

Mean
2487
2933
3561
3598

The data given as peak areas in Tab. 2 show that samples with larger particles result in a
lower extraction yield of the analyte (quinine). A typical chromatogram is shown in Fig. 3.
The relative quantitative ratio between the four major alkaloids was constant in all samples
measured.
When grinding samples, an increase in temperature in the sample may occur due to
friction energy. Therefore, the temperature of the grinding wheels as well as the sample
was measured before and after grinding. In all cases, no change in temperature within
1C was detected (data not given). Sieving analysis was performed on sample 4 from
Table 2 and on Rhei radix and Liquiritiae radix after grinding. The results are given in

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S. H. Hansen et al.:

Table 3. A factor that may influence the fineness of the powder obtained is the degree of
dryness of the sample.

Fig. 3.

Chromatogram of an extract of Cinchona bark.

1) Cinchonine, 2) cinchonidine, 3) quinidine, and 4) quinine

Tab. 3.

Sieving analysis of three samples after grinding

Sample
Cinchona bark
Rhei radix
Liquiritiae radix

% passed
sieve no 355 sieve no 180
73.5
30.8
93.6
57.6
93.7
50.5

HPLC
An HPLC system for the separation of the four major alkaloids (quinine, quinidine, cinchonine, cinchonidine) in Cinchona bark as well as other constituents was developed. It was
important that the system would also be compatible with mass spectrometric detection as
this allows for better identification of alkaloids than UV. For HPLC, a modern solid core
C18 column was chosen as it is known to deliver good peak symmetries. Acetonitrile was
used as the organic modifier in most of the reversed-phase system for the separation of
the Cinchona alkaloids published until now, giving a fair separation of all four major
alkaloids [47]. However, a few experiments showed that the addition of methanol to the
mobile phase improved the resolution between all four major alkaloids and thus, a mixture
of acetonitrile + methanol (40:60 v/v) was used. The pH of the mobile phase was kept at
4.0 using 20 mM ammonium formate adjusted with formic acid. To be sure that all
constituents were eluted from the column, gradient elution was performed. Detection was
performed using UV at 330 nm where the major Cinchona alkaloids have an absorption
maximum. Although this is not the strongest absorption, the choice of the longer
wavelength improves the detection selectivity.

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A Robust and Inexpensive Grinding Device for Herbal Samples

373

Experimental
Chemicals and Reagents
Cinchona cortex (Ph.Eur. quality) was obtained from Alfred Galke GmbH, Gittelde/Harz,
Germany. Rhei radix was a gift from Naturmedicinsk Museum, University of Copenhagen
and organic Liquiritiae radix was obtained from Naturdrogeriet A/S, Hrning, Denmark.
Quinine sulfate was obtained from Merck (Darmstadt, Germany). All other chemicals were
of HPLC or analytical grade quality and were obtained from Merck.
The Grinding Device
A commercially available coffee grinder (Rancilio Rocky) was purchased from Rigtig Kaffe
A/S, Skanderborg, Denmark (price in Denmark about 250 EUR). It was rebuilt in the
following way in order to reduce the internal dead volume and to ease the assembling/
disassembling of the grinding machine: an insertable alumina block (Figure 1.5 and
Figure 2) was manufactured to fill out the dead volume around the lower cutter inside the
grinder. The manufacture of the insert according to Figure 2 took about 23 hours by a
precision mechanic for a cost of materials of about 3040 EUR.

2
1

8
Fig. 1.

1) The purchased coffee grinder; 2) The grinder with the lower grinding wheel
observe the dead volume; 3) The lower grinding wheel; 4) The coffee grinder
fully disassembled; 5) Zero dead volume insert; 6) Lower grinding wheel
inserted in the zero dead volume unit; 7) Upper grinding wheel assembled; 8)
The modified grinder dissembled; and 9) The modified grinding device

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S. H. Hansen et al.:

In the upper cutter wheel, a pin is inserted to ease the assembling/disassembling of the
cutter in order to be able to clean the inside. For sample collection, a stainless steel chute
is inserted into the outlet with zero dead volume.
A technical drawing of the zero dead volume insert is shown in Figure 2.

Fig. 2.

Technical drawing of the zero dead volume insert

HPLC
The HPLC system consisted of an Agilent 1200 system consisting of an on-line degasser,
a binary pump G1312B, an autosampler G1367C, a column oven G1316B, and a DAD
detector G1315C. The analytical separation column was a Kinetex XB-C18 (150 x 2.1 mm)
with 2.6 m particles. The gradient elution was performed using mobile phase A: 0.2 M
ammonium formate buffer pH 4.0 and water (10:90 v/v) and mobile phase B: acetonitrile
and methanol (40:60 v/v) at a flow rate of 0.2 ml/min. The gradient was 18% B from 0-10
min, then changed from 18% B to 36% B from 10-25 min returning to 18% B after 26 min
with a total run time of 35 min. For UV detection, the wavelength at 330 nm was used.

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A Robust and Inexpensive Grinding Device for Herbal Samples

375

Sample Preparation for Grinding

Typically, the bark samples are cut into pieces the size of a coffee bean or smaller, and
are transferred to the grinder. The fineness of the powder to be produced is determined by
the tunable upper cutter. The grinding process takes typically less than 10 seconds.
Sample Preparation for HPLC
One hundred mg of ground Cinchona bark was added to 2.0 mL of 70% methanol
containing 0.1% formic acid. The mixture was ultrasonicated for 10 min and then diluted to
10.0 mL with mobile phase A. After centrifugation at 10,000 g, 5 l was injected into the
HPLC.
Particle Size and Temperature Measurements
The particle size was measured on a Mastersizer 2000, Malvern Instruments,
Worcestershire, UK. The temperature measurements were performed using a FLUKE, 62
Mini IR thermometer, Optris, RS Components A/S, Copenhagen, Denmark.

Conclusion
An efficient and inexpensive grinding device for the disintegration of herbals has been
constructed on the basis of a commercially available coffee grinder.
Very fine and homogenous powder can be obtained gently and with a uniform and
repeatable particle size, which enables repeatable quantitative data even when using very
small sample sizes. It is important to note that no increase in temperature was observed
due to the very short grinding time in seconds. Furthermore, a new LC method for
fingerprinting Cinchona bark is presented.

Acknowledgement
The authors wish to thank Lasse Johansson for the help with the mechanical modification
of the coffee grinder. The research presented in this publication was supported by a grant
from the Carlsberg Foundation, Denmark.

Authors Statement
Competing Interests
The authors declare no conflict of interest.

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